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OPEN Termite’s Twisted Mandible Presents Fast, Powerful, and Precise Strikes Kuan-Chih Kuan1,5, Chun-I Chiu1,5, Ming-Chih Shih2, Kai-Jung Chi2,3,4 ✉ & Hou-Feng Li1 ✉

The asymmetric mandibles of termites are hypothetically more efcient, rapid, and powerful than the symmetric mandibles of snap-jaw or termites. We investigated the velocity, force, precision, and defensive performance of the asymmetric mandibular snaps of a termite species, Pericapritermes nitobei. Ultrahigh-speed recordings of termites revealed a new record in biological movement, with a peak linear velocity of 89.7–132.4 m/s within 8.68 μs after snapping, which caused an impact force of 105.8–156.2 mN. High-speed video recordings of ball-strike experiments on termites were analysed using the principle of energy conservation; the left mandibles precisely hit metal balls at the left-to- front side with a maximum linear velocity of 80.3 ± 15.9 m/s (44.0–107.7 m/s) and an impact force of 94.7 ± 18.8 mN (51.9–127.1 mN). In experimental fghts between termites and predators, Pe. nitobei killed 90–100% of the generalist ants with a single snap and was less likely to harm specialist ponerine ants. Compared with other forms, the asymmetric snapping mandibles of Pe. nitobei required less elastic energy to achieve high velocity. Moreover, the ability of P. nitobei to strike its target at the front side is advantageous for defence in tunnels.

Some invertebrates’ elastic power-amplifying systems that incorporate latches and springs can overcome the physiological limits of muscle contraction and perform a powerful or rapid movement1,2. For example, mantis shrimps (Crustacea: Stomatopoda) perform high-speed strikes to prey by using elastic energy stored in their raptorial appendages3. Springtails (Hexapoda: Collembola) escape from enemies with quick and powerful jumps by using their springing organ4. Many ants and termites, such as the snap-jaw ant Mystrium camillae Emery (: Formicidae)5 and the soldiers of termite Termes panamaensis (Snyder) (Blattodea: Termitidae)6, have mandibles that are morphologically specialised for powerful snapping attacks. Te snapping speeds of the mandibular attacks of M. camillae (111.1 m/s)5 and T. panamaensis (67 m/s)6 are the most rapid move- ments currently reported1,5,6, followed by the mandible-closing movement of bauri Emery trap-jaw ants (64.3 m/s)7, the diving of gyrfalcons (58 m/s)8, the nematocyst discharge of jellyfsh (37 m/s)9, and strike behaviour of mantis shrimps (31 m/s)1. Te snapping mandibles of termite soldiers have two forms: symmetric and asymmetric10,11. T. panamaensis has two narrow and elongated symmetric snapping mandibles that press together until they slide against each other (Fig. 1a). Tese mandibles strike enemies along their lateral sides6. By contrast, the asymmetric snapping mandibles of some termites are relatively short and wide, with a twisted lef mandible and curved right mandi- ble10. Te right mandible presses against the lef until the two surfaces slide. Moreover, the lef mandible snaps in a clockwise motion (Fig. 1b), presumably striking enemies along the front side. Additionally, asymmetric snapping mandibles may perform more violent strikes than symmetric mandibles can by storing elastic energy in the lef twisted mandible12. However, no conclusive evidence has supported these hypotheses. Ants are major natural predators of termites. Predator ants include specialist predators such as Pachycondyla spp. (Formicidae: Ponerinae)13 and generalist predators such as Anoplolepis spp. and Pheidole spp.14,15. Te power- ful mandibular snapping of termite soldiers is hypothesised to be a specialised defence mechanism against ants16. Termites can snap the ground to leap away from ants17,18 or snap at ants to push them away16 or even kill them6. However, these defensive behaviours have not been studied in termites with asymmetric mandibles.

1Department of Entomology, National Chung Hsing University, Taichung City, Taiwan. 2Department of Physics and Institute of Biophysics, National Chung Hsing University, Taichung City, Taiwan. 3Department of Life Sciences, National Chung Hsing University, Taichung City, Taiwan. 4The iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung City, Taiwan. 5These authors contributed equally: Kuan-Chih Kuan and Chun-I Chiu. ✉e-mail: [email protected]; [email protected]

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Figure 1. Snapping mandibles of termite soldiers. (a) In the symmetric snapping of Termes panamaensi6,10, elastic energy is stored in both mandibles, as indicated by the deformation and motion of the lef (L) and right (R) mandibles. (b) In the asymmetric snapping of Pericapritermes spp.10, elastic energy is stored in only the lef mandible when its anterior part rotates about the joint or pivot point O. Te posterior part remains stationary during snapping. (c) Morphology of the twisted lef mandible of the Pericapritermes nitobei termite used in this study. Te length of the anterior part of the lef mandible (LA) and the mass of its subsections A1 and A2 (i.e., MA1, MA2) are required to estimate its moment of inertia (IA). Te subsections A1 and A2 were prepared by performing two cuts; one cut was made at the pivot point (C1) and the other was made at the centre of the anterior part (C2).

Tis study investigated the velocity, force, precision, and defensive behaviours of asymmetric mandibu- lar snaps performed by Pericapritermes nitobei (Shiraki) soldiers. Specifcally, we conducted ultrahigh- and high-speed video recordings, ball-strike experiments, and ant-defence assessments to test the following hypoth- eses: (1) snaps of asymmetric mandibles are more rapid and powerful than snaps of symmetric mandibles are, (2) asymmetric mandibles can precisely strike at the front side, and (3) asymmetric mandibles are efective for defending against ants. In addition to revealing the fastest known animal movement, we compared asymmetric and symmetric termite mandibles and analysed the evolution and mechanical efects of mandible morphology. Methods Termites and ants. A total of 100 Pe. nitobei termite soldiers were collected by excavating the soil beneath stones and logs in three locations in Taiwan, namely Xiaping Tropical Botanical Garden (23.77°N, 120.67°E), Huisun Forest Station (24.09°N, 121.03°E), and Dakeng (24.19°N, 120.79°E) (Supplementary Information, Table S1). Pe. nitobei is a soil-feeding termite that does not have a centralised nest; they distribute their eggs and larvae in subterranean tunnels beneath stones19. Tey generally forage on organic matter adjacent to plant roots and rarely appear on the ground surface19. Because Pe. nitobei colonies are difcult to maintain in laboratory conditions, all experiments were conducted within 1 day afer collection. Te sample sizes and collection sites of subjects used in all the experiments are summarised in Table S1 of the Supplementary Information. Natural predators of Pe. nitobei were identifed on the basis of references and feld observations. Pheidole and Anoplolepis spp. are generalist termite predators14,15, and Anochetus and Pachycondyla spp. are specialist predators of termites that perform specialised striking behaviours on termite soldiers or recognise termite odours and raid termite nests20,21. In this study, Ph. megacephala (Formicidae: Myrmecinae) and Anop. gracilipes (Formicidae: Formicinae) were assumed to be generalist predators of Pe. nitobei. Both species have been observed to for- age on the ground and prey on termites in Xiaping Tropical Botanical Garden22,23. Two ponerine ant species (Formicidae: Ponerinae), namely Anochetus taiwaniensis and Pa. javanus, were assumed to be specialist predators of Pe. nitobei. Anoc. taiwaniensis was observed foraging for termites in rotting logs at Huisun Forest Station. Pa. javanus was observed foraging in the soil surrounding the subterranean galleries of Pe. nitobei in Xiaping Tropical Botanical Garden, Huisun Forest Station, and Dakeng. Te generalist and specialist ants used in the assessments were collected from National Chung Hsing University and Huisun Forest Station, respectively (Supplementary Information, Table S1).

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Estimating velocity and force by using an ultrahigh-speed camera. In each experiment, a termite was placed in the centre of a plastic petri dish (diameter: 90 mm) with a flter paper (Advantec No. 1, Toyo Inc.; diameter: 90 mm). Snapping behaviour was then recorded at 460,830 frames per second (fps) by using an ultrahigh-speed video camera (Model: v2512, Phantom, New Jersey, USA) with a resolution of 128 × 128 pixels2. Te linear velocity of the mandible tip (VMT) was calculated as follows:

VLMT = AAω , (1)

where LA and ωA are the length and angular velocity of the anterior rotating part about the pivot point O (Fig. 1c), respectively. Te parameter ωA was calculated from the video footage by using Phantom CV 2.8 (Phantom, New Jersey, USA) for image analysis. 6,7 Te snapping force (FA) was calculated using the following equation: 1 FMAA= LAAα , 3 (2)

where MA and αA are the mass and angular acceleration of the anterior rotating part of the lef mandible, respec- tively. Te average αA can be calculated as follows:

ωA αA = , tA (3)

where tA is the time required to reach the maximum ωA. Te snapping energy (EA) (i.e., the kinetic energy due to rotation) was calculated as follows:

1 2 EIAA= ωA 2 (4)

where IA is the moment of inertia of the anterior rotating part about the pivot point O (Fig. 1c). Because the ter- mite’s lef mandible cannot be modelled using a common geometrical form, we adjusted the moment of inertia (IA) by using a constant K as follows:

2 IKAA=.MLA (5)

Te parameter K was calculated using the location of the anterior lef mandible’s centre of mass (LCOM), length of the anterior mandible part (LA), and mass of mandible parts A1 (MA1) and A2 (MA2) (Fig. 1c; Table S3) as follows:

2 2 M L  M LL+  K =⋅A2  COM  +⋅A1  COMA ,     MA  22LA  MA  LA  (6)

and LCOM was calculated as follows:

1/43LMAA21+ /4LMAA LCOM = . MA (7) Te termite’s lef mandible is morphologically inhomogeneous. Te lef mandible is wide in the posterior part and at the pivot point and narrow at the centre of the anterior part (Fig. 1c). To obtain parts A1 and A2, we used a scalpel blade to cut the lef mandible frst at its pivot point (C1) and then in the middle of the anterior part (C2) (Fig. 1c). To avoid measurement errors, MA1 and MA2 were calculated as the means of 12 A1 and 14 A2 parts, respectively. Te total mass of the anterior lef mandible (MA) was the sum of MA1 and MA2. A total of 17 soldiers were used to measure the mass and length of the lef mandible (Table S1). Images were captured using a Leica M205 C stereomicroscope with a Leica MC170 HD digital camera (Leica Microsystems, Wetzlar, Germany), and all lengths were calculated using the LAS Image Analysis software module (Leica Application Suite V4.4.0, Leica Microsystems, Wetzlar, Germany). Te mass of the termites was measured using an analytical balance (AG245, Mettler Toledo, Greifensee, Switzerland). Termite behaviours were considered abnormal in ultrahigh-speed recordings under the following conditions: (1) three of fve termites died in the petri dish before snapping, (2) the other two termites snapped without being triggered and did not extend their antennae forward to detect targets in front of them, and (3) the termites died immediately afer snapping once. We assumed that the bright light required for ultra-speed recording (brightness of approximately 85,000 lux) caused these abnormal behaviours. Terefore, we used a high-speed video camera with less intense light (approximately 8,670 lux) to observe the snapping behaviour and reassess the snapping velocity and force. To minimise the efects of light on ant and termite behaviour, we recorded fghts between ter- mites and ants by using a smartphone camera with a light intensity of approximately 850 lux.

Estimating mandible strike accuracy, velocity, and force by using a high-speed camera. Each termite was placed in a plastic petri dish with a filter paper, as in the ultrahigh-speed recordings. Mandible-snapping behaviour was triggered by touching a termite’s antennae from the front by using tweezers. Snapping was recorded at 1,000 fps with a high-speed video camera (MotionPro X3, Integrated Design Tools, Inc., Tallahassee, FL, USA). Behavioural phases were identifed, and the duration of each phase was measured using the recorded images.

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To measure the striking angle and reassess the velocity and force of mandible snapping, we conducted ball-strike experiments on 15 soldiers. In each experiment, a metal ball (2.25 mg, 0.7 mm in diameter) with a mass similar to that of the termite was obtained from the tip of a ballpoint pen (TOWO OP-100, 0.7 mm) and placed in front of the soldier’s head. Te soldiers snapped at the ball 1–2 seconds afer it was placed. When the lef mandible hit the metal ball, the termite soldier and the ball moved away from each other; movements were recorded by the aforementioned high-speed video camera at 1,000 fps. Te diameter of the petri dish was used as a scale for distance measurement. Te experiment was repeated until the termite was exhausted and unresponsive to stimuli. For each termite, 3–14 snaps were recorded. Assuming that no energy loss caused by air resistance or ball rotation occurred, according to the energy con- servation principle, the kinetic energy of the rotating anterior part of the lef mandible (EA) on striking the ball equals the sum of the linear and angular kinetic energy of the ball and termite as follows:

1 221 1 221 EIAA==ωωABMVBT++MVTTI T , 2 2 2 2 (8)

where MB and MT are the masses of the metal ball (2.25 mg) and termite, respectively; VB and VT are the linear velocities of the ball and termite, respectively; ωT is the angular velocity of the termite; and IT is the moment of inertia of the termite. Te parameters VB, VT, and ωT were measured according to the motions of the termite and ball by using video analysis sofware (Tracker 4.91, http://physlets.org/tracker/). Te parameters VT and VB were calculated from the linear displacement of the termite and ball between the frst two frames afer the termite struck the ball. Te variable ωT was calculated using the termite’s number of rotations over a specifc time period. By assuming the termite to resemble a cylinder rotating about its centre of mass, its IT was calculated from its 24 mass (MT) and body length (LT) as follows:

1 2 IMTT=.LT 12 (9)

Subsequently, the snapping force (FA) could be calculated using Eqs. (2) and (3). Te linear velocity of the tip of the snapping lef mandible on striking the ball (VMT) could be calculated using Eqs. (1), (4), and (5) as follows:

2EA VLMT ==AAω , MKA (10)

where K is a constant for adjusting the moment of inertia (IA) and MA is the total mass of the anterior lef mandi- ble. Te parameter K was calculated using Eqs. 6 and 7, and EA was calculated using Eq. 8. We evaluated the precision of mandibular snaps by variations in the ball’s movement directions afer a snap. Precise snaps occurred in a particular hitting zone with less variation in movement directions. Te ball’s move- ment directions were calculated as the angle of deviation from the termite’s body axis. To determine whether the snaps had a particular hitting zone, we performed a Shapiro–Wilk test for confrming the normality of angle measurements.

Assessment of termites defending against ants. To observe snapping behaviour against predators, termite soldiers were forced to fght with workers of Anop. gracilipes, Anoc. taiwaniensis, and Pa. javanus and minor workers of Ph. megacephala. Te collection locations of ants and the number of termites used in each trial are presented in Table S1. Fights occurred in a petri dish (diameter: 55 mm) layered with a flter paper (Advantec No. 2, Toyo Inc.; diameter: 55 mm) containing 1 mL of water. In each trial, four termites and four ants of the same species were placed on the flter paper, and their fghts were recorded at 30 fps for 15 minutes on a smartphone camera (ZenFone 3 ZE520KL, ASUSTeK Computer Inc.). We reconfrmed whether the ants were predators of Pe. nitobei according to the number of encounters with termites and the number of attacks performed by each ant species. To evaluate the termites’ defensive performance, the numbers of mandibular snaps and hits as well as ants killed were recorded. Six trials were performed for each ant species, and the data from all trials were combined to calculate the ants’ attack rates and termites’ hitting and killing probabilities. Te attack rate of ants was the ratio of the number of attacks performed to the number of encounters with termites. For termites, the hitting probability was the ratio of the number of hits to the number of snaps and the killing probability was the ratio of the number of ants killed to the number of hits. Pairwise comparisons for hitting and killing probabilities were conducted using Fisher’s exact tests with Bonferroni corrections. All the statistical data were obtained using R sofware (v. 3.3.1, R Development Core Team, 2013)25. Results Mandible-snapping sequences. Figure 2a displays a behaviour schematic. In the high- and ultra- high-speed recordings, the mandible-snapping behaviour of Pe. nitobei soldiers began with the right mandible pressed against the lef mandible to store elastic energy (loading phase). Te termites required 261.3 ± 43.1 ms (n = 10) to complete the loading phase: During the frst 213.8 ± 43.5 ms, both antennae extended forward as the right mandible continued to press against the lef mandible; it took another 47.5 ± 22.5 ms to turn their antennae towards their rear before snap. Mandibular sliding was completed in 21.7–43.4 μs (Fig. 2b,c).

Velocity and force of mandible snaps. In the ultrahigh-speed recordings of mandible snapping, the peak linear velocity occurred at 8.68 μs (tA) afer execution and had values of 132.4 (Fig. 2b; Supplementary Movie S1) and 89.7 m/s (Fig. 2c; Supplementary Movie S2) for two termites. Te peak snapping energy of these termites was 41.4 and 19.0 μJ respectively, and their peak snapping forces were 156.2 and 105.8 mN, respectively.

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Figure 2. Behavioural phases of Pericapritermes nitobei mandibular snaps. (a) Stages 1–3: High-speed recordings from 10 soldiers at 1,000 frames per second (fps) indicated that the right mandible pressed against the lef mandible for 261 ± 43 ms before the mandibles slid across each other to snap, and termites raised their antennae 47.5 ± 22.5 ms before the snap (Stages 2–3). Stage 3–4: Single snap of two soldiers (b and c) recorded by an ultrahigh-speed video camera at 460,830 fps. Mandibular snaps were performed over 21.7–43.4 μs. Te peak linear velocity (VMT) of two soldiers at 8.68 μs were 132.4 and 89.7 m/s. Images in (b,c) were obtained by transforming Supplementary Movies S1 and S2, respectively, to frames using codes written in Python language (v. 3.8) with the package opencv-python.

In the ball-strike experiments, the energy from the termite’s mandible snap caused the termite and metal ball to move away from each other and sometimes rotate (Fig. 3a, Supplementary Movie S3). On the basis of size and velocity measurements of 15 termite soldiers in 92 events (Tables S2, S3, Supplementary Information), the snapping energy (EA) had a mean of 9.5 ± 3.2 μJ and maximum of 15.8 ± 5.9 μJ (range: 4.6–27.4 μJ). Te mean and maximum linear velocities of the mandible tip on striking the ball (VMT) were 60.4 ± 12.0 and 80.3 ± 15.9 m/s (range: 44.0–107.7 m/s), respectively (Fig. 3b). Assuming that the lef mandible struck the ball at its maximum lin- ear velocity (tA) 8.68 μs afer snapping (Fig. 2b,c), the mean and maximum snapping forces (FA) were 71.3 ± 14.1 and 94.7 ± 18.8 mN (range: 51.9–127.1 mN), respectively (Fig. 3c). Te body measurement and snap performance data are summarised in Table 1.

Striking angle of mandible snaps. In the ball-strike experiments, the movement directions of the ball afer being snapped by the Pe. nitobei mandible ranged from 2.3° to 122.1° and were normally distributed (Fig. 3d) (Shapiro–Wilk test: W = 0.99, p = 0.70) with a mean of 52.9° ± 22.7°. Tis result suggests that termites with asymmetric mandibles strike most enemies at the front-lef side.

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Figure 3. Snap performance of the Pericapritermes nitobei soldier’s asymmetric mandible in ball-strike experiments (92 events from 15 termites, A–O). (a) Motion of the termites and metal ball. When the lef mandible hit the metal ball, the termite soldier and ball moved away from each other. Yellow and blue arrows indicate the position of the termite and metal ball, respectively. (b) Linear velocity VMT. (c) Snapping force FA. Te maximum recorded VMT and FA values for each soldier are displayed above each bar. (d) Movement directions of the metal ball. Each blue line indicates a record of one moving metal ball. Angle normality was assessed using the Shapiro–Wilk test. Images in (a) were obtained by transforming Supplementary Movie S3 to frames using codes written in Python language (v. 3.8) with the package opencv-python.

Ant-defence performance. Te generalist ants, namely Ph. megacephala and Anop. gracilipes, encountered termites 93 and 107 times and attacked termites 8 (8.6%) and 18 (17.1%) times in the defence experiments, respectively. Te specialist ants, namely Anoc. taiwaniensis and Pa. javanus, encountered termites 300 and 143 times and attacked termites 102 (34.0%) and 33 (23.1%) times, respectively. Te results indicated that Ph. meg- acephala and Anop. gracilipes are facultative or generalist predators of termites. Moreover, compared with Ph. megacephala and Anop. gracilipes, Anoc. taiwaniensis and Pa. javanus exhibited higher levels of aggression, which is consistent with them being specialist predators of termites. Te termite soldiers had a signifcantly high probability of hitting Ph. megacephala (83%), signifcantly low probabilities of hitting Anop. gracilipes (13%) and Anoc. taiwaniensis (30%), and a moderate probability (41%) of hitting Pa. javanus (Fig. 4a) with a mandible snap. Te mandibular snaps of termites killed most generalist ants in a single hit (mortality: 90–100%, Fig. 4b). By contrast, none of the ponerine ants were killed by a mandibular snap (Fig. 4b). Only 2 of 24 Anoc. taiwaniensis (8.3%) individuals were had immobilised lef mandibles afer being snapped by a termite.

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Snapping termites Trap-jaw ant Snap-jaw ant Mystrium camillaed Pericapritermes Termes Odontomachus Major Minor nitobeia panamaensib bauric worker worker Filming rate (kfps) 1 460 40 50 480 480 Min. 2.91 — 12.10 — —

MT (mg) Max. 3.39 — 14.90 — — Mean 3.22 1.76 — 2.66 0.48 Min. 5.90 — — — —

LT (mm) Max. 6.20 — — — — Mean 6.06 5.00 — — — Min. — — 111.0 — —

Body measurements MA (μg) Max. — — 145.0 — — Mean 28.0 30.0 — 85.9 8.5 Min. 0.97 — 1.24 — —

LA (mm) Max. 1.12 — 1.38 — — Mean 1.06 1.50 — 1.56 0.84 Min. — — — — — –14 2 IA (×10 kg·m ) Max. — — — — — Mean 0.56 2.25 — 6.9 0.2 Min. — 8.68 — — — —

tA (μs) Max. — 8.68 — — — — Mean — 8.68 25.0 130.0 34.8 14.6 Min. 17.5 89.7 — 35.5 — —

VMT (m/s), peak Max. 107.7 132.4 67.0 64.3 107.4 111.1 Mean 60.4 111.1 56.0 — 86.9 93.3 Min. — 10.2 — 2.8 — — 4 ωA (×10 rad/s) peak Max. — 15.3 — 4.7 — — Mean — 12.8 — — 5.6 11.1 Min. — 11.7 — — — — 9 2 Snap Performance αA (×10 rad/s ) peak Max. — 17.6 — — — — Mean — 14.7 — — 6.0 17.2 Min. 4.6 19.0 — — — —

EA (μJ) peak Max. 27.4 41.4 — — — — Mean 9.5 30.2 15.0 — 109.0 12.4 Min. 20.4 105.8 — — — —

FA (mN) peak Max. 127.1 156.2 — 69 — — Mean 71.3 131.0 54 47 268 41 Min. 1.6 3.9 — 0.4 — — 3 FA/body weight (×10 BW) Max. 3.9 4.6 — 0.5 — — Mean 3.0 4.2 3.1 — 10.3 8.7

Table 1. Comparison of body measurements and snap performance between the snapping termites and ants. Body measurements: MT, body mass; LT, body length; MA, mass of the waving part of lef mandible; LA, length of the waving part of lef mandible; IA, the moment of inertia of waving part of the lef mandible. Snap performance: tA, duration of mandible strike; VMT, velocity of the mandible tip; ωA, angular velocity of waving part of the lef mandible; αA, angular acceleration of mandible strike (calculated as αA = ωA/tA); EA, energy released by mandible strike; FA, force caused by mandible acceleration; FA/body weight, ratio of maximum striking force and body weight. adata from current study (see Tables S1, S2, and S3 in Electronic Supplementary Material); bdata from Seid et al.6; cdata from Patek et al.7; ddata from Larabee et al.5.

Discussion The most rapid animal movement. Te most rapid recorded animal movement has been updated several times. Te cheetah was considered to be the fastest animal before 199826. Te newest record was set by the snap- jaw ant M. camillae, whose mandible can achieve a maximum velocity of 93 ± 14.5 m/s5. Our results revealed that the snapping movement speed of Pe. nitobei soldiers’ asymmetric mandibles was similar to or even more rapid than that of snap-jaw ants’ symmetric mandible. Ultrahigh-speed recordings of snap-jaw ants indicated that 3 of 10 individuals had snapping velocities greater than 100.0 m/s, and the maximum recorded velocity was 111.1 m/s5. In our study of Pe. nitobei, the maximum snapping velocity calculated in the ball-strike experiment was 107.7 m/s, which was a conservative estimate because we disregarded the rotational energy of the metal ball

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Figure 4. Defensive performance of mandibular snaps by probability (values above each bar) of (a) hitting and (b) killing four predator ant species. Bars with identical letters were not signifcantly diferent at p < 0.05 (Fisher’s exact tests with Bonferroni corrections). Termites could not kill their specialist predators Anochetus taiwaniensis and Pachycondyla javanus.

and assumed no energy loss due to friction. Te peak snapping velocity of Pe. nitobei calculated using ultra- high-speed recordings reached 132.4 m/s, which was twice the velocity of the symmetric mandibles of T. pana- maensis (67 m/s)6 and sets a new record for the most rapid biological movement.

Powerful and efcient snapping mechanism of the asymmetric mandible. Te ‘snap-jaw’ mech- anism, which combines a spring and tool in one appendage, has been proposed to be more efcient than most other power-amplifed appendages with distinct structures2,5. Te peak impact force generated by a Pe. nitobei soldier may be approximately 4,600 times their body weight (BW), which is within the range estimated for other snap-jaw systems (3,000–10,000 BW). Te aforementioned force is one order of magnitude higher than that reported for trap-jaw ants (approximately 500 BW)7 (Table 1). Compared with symmetric mandibles, asymmetric mandibles require less energy to achieve super-rapid movement (Table 1). Te average snapping velocity of Pe. nitobei soldiers calculated in the ball-strike experiment 6 was similar to that reported for T. panamaensis (60.4 m/s vs. 56 m/s); however, the required elastic energy (EA) stored in the Pe. nitobei mandible was only two-thirds that stored in the T. panamaensis mandible (9.5 μJ vs. 15.0 μJ)6 even though Pe. nitobei has a body size twice that of T. panamaensis. At the peak snapping velocity of 132.4 m/s, Pe. nitobei soldiers must store 41.4 μJ of elastic energy, which is less than half of the 109 μJ of elastic

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energy required by major workers of M. camillae with similar body size and lower snapping velocity (111.1 m/s). Table 1 indicates that the lower energy requirement of Pe. nitobei asymmetric mandibles for achieving super-rapid movement can be attributed to their shorter rotating section (LA) and consequently smaller moment of inertia (IA) than those of symmetric mandibles. Elastic energy is stored in the deformed shafs of both the symmetric snapping mandibles of T. panamaensis and the snap-jaw ant M. camillae (Fig. 1a)5,6, whereas energy storage in the asymmetric mandibles of Pe. nitobei is confned to the bendable ‘joint’ (or ‘pivot’ in Fig. 1c) at the curved part of the lef mandible. Te lef mandible of a Pe. nitobei soldier is complex in shape, and this mandible is generally laterally compressed but wide along the dorsal–ventral axis (Fig. 1b,c). Te location of minimum lateral thickness creates a ‘joint’ in the continuous and single-segmented mandible. Te mandible base and its posterior part are more robust than its anterior coun- terpart is. Assuming homogeneous material composition, the anterior part of the mandible can rotate about the ‘joint’ by lateral pressure from the right mandible. Te lef mandible twists towards its tip to become laterally wide. Consequently, the lef mandible tip becomes more difcult to bend and can efectively transfer impact energy for defence. We suggest that because of its morphological modifcations, the twisted lef mandible of a Pe. nitobei soldier has a functional joint for storing elastic energy, which creates a shorter rotating section that requires less stored elastic energy to achieve greater velocity. In other words, asymmetric snapping mandibles are more efcient than symmetric snapping mandibles are.

Antenna extension as an aiming behaviour. Because the direction of movement is unlikely to be cor- rected during high-speed ballistic movement, an aiming or triggering mechanism before initiating movement is crucial for precision27,28. For example, the archer fsh Toxotes jaculatrix can identify the three-dimensional location of their target and precisely aim at and shoot it28. Te snapping of a Mystrium snap-jaw ant’s man- dible is triggered by sensory hairs on its labrum to ensure precise timing for catching rapidly moving prey27. Te Odontomachus trap-jaw ant touches its prey using its elongated antennae before snapping, which may be a type of aiming behaviour29. We observed that Pe. nitobei had a specifc striking angle of 52.9° ± 22.7°, and it could hit active ants with a moderate probability of 8.6–34.0%. Tese results implied that Pe. nitobei aims before snapping to adjust its strik- ing angle and distance from the target. We suggest that the forward extension of antennae observed in termite soldiers is an aiming behaviour and that the backward movement of the antennae beginning 45.9 ± 10.1 ms before snapping is a synergic behaviour (Fig. 2a). Because the snapping termite soldiers of Pe. nitobei lack eyes30, they likely locate enemies by touching with their antennae instead of by vision, as observed in Odontomachus trap-jaw ants29 and T. panamaensis6. Tis hypothesis was supported by the observation that the snapping behaviour of Pe. nitobei was triggered by touching their antennae tips with tweezers. However, forward-extended antennae may be at risk of being cut by the rapidly moving lef mandible. Terefore, moving the antennae backward immediately before snapping can reduce this risk.

Functional adaptation of asymmetric mandible snapping. Te defensive strategies of termite sol- diers are highly diversifed and adapted to their habitats10,31. For example, Cryptotermes termite soldiers that live in single pieces of wood or dead tree branches use their heavy phragmotic heads to block enemies12. Nasutitermes termite soldiers, which construct arboreal nests and forage in trees, eject viscous secretions to defend the for- aging party from ant attacks32,33. Biting or slashing mandibles are most commonly observed among primitive wood-feeding termites, such as most kalotermitids and rhinotermitids34,35. Snapping mandibles have evolved in four independent clades of Termitidae and in one clade of Kalotermitidae10,34,36,37. Tese mandibles have been hypothesised to be a defensive weapon, especially in tun- nels6,12, where both termites and their enemies are confned to narrow spaces (e.g., approximately 2-mm wide, as reported for Pe. nitobei and Sinocapritermes mushae19,38). Narrow tunnels limit termite movement as well as the gape of biting or slashing mandibles, thereby reducing their defensive performance. Tis study demonstrated that termites with asymmetric snapping mandibles strike at the front-lef side without moving forward or opening their mandible wide. We suggest that asymmetric snapping mandibles are more advantageous than symmetric mandibles are when termites fght with intruding ants in tunnels. Te hypothesised function of termite mandibular snaps is to kill or push out ants6,16. We observed that the defensive function of Pe. nitobei mandibular snaps varied with the encountered ant species. Mandibular snaps could kill generalist predators but barely harmed specialist predators, particularly Pa. javanus, which is observed in most subterranean gallery systems of Pe. nitobei in the feld (4/5 localities, unpublished data) and is likely the main predator of Pe. nitobei. Pa. javanus may be able to resist mandibular snaps for the following reasons: (1) Pa. javanus is larger than other ants (head width of approximately 2.5 mm for Pa. javanus vs. approximately 0.6–1.5 mm for other species); (2) the exoskeleton of Pa. javanus is thicker than that of most generalist and pon- erine ants;39 and (3) Pa. javanus initiated long-distance attacking behaviour approximately 6.8 mm away from the mandible tip of Pe. nitobei (Fig. 5d), which was considerably longer than the attacking distances observed in Ph. megacephala (approximately 0.7 mm, Fig. 5a), Anop. gracilipes (approximately 0.6 mm, Fig. 5b), and Anoc. taiwaniensis (approximately 1.4 mm, Fig. 5c). Moreover, we observed that Pa. javanus could clamp the termite’s mandibles (Fig. 5d) to prevent counterattacks. Tus, we hypothesised that the aforementioned characteristics and behaviours of Pa. javanus were selected to withstand the stress of termites’ defensive behaviours, particularly powerful mandibular snaps. We also propose that the snapping mandible of Pe. nitobei can function as weapon to defend against their generalist but not specialist ant predators. In conclusion, we provided evidence that the asymmetric snapping mandibles of Pe. nitobei are a rapid, pow- erful, and precise ballistic weapon. Asymmetric snapping behaviour was particularly advantageous in defending against intruding ants by striking them at their front side in subterranean tunnels.

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Figure 5. Attack behaviours of (a) Pheidole megacephala, (b) Anoplolepis gracilipes, (c) Anoc. taiwaniensis, and (d) Pa. javanus on Pe. nitobei. Te attacks of Ph. megacephala, Anop. gracilipes, and Anoc. taiwaniensis were initiated afer their antennae touched the mandibles or head of Pe. nitobei (0 ms), with a short distance between the heads of these three species and the mandible tip of Pe. nitobei (approximately 0.6–1.5 mm). By contrast, Pa. javanus initiated an attack when its head was approximately 6.8 mm away from the mandible tip of Pe. nitobei (0 ms). Tis attack was followed by rapid movement toward the termite’s head (33.3 ms) and clamping at its mandibles (66.6 ms).

Data availability Ultrahigh-speed video recordings are available as Supplementary Movies S1 and S2. Termite measurements are provided in the Supplementary Information. Video recordings and datasets of the ball-strike experiments and fghts between termites and ants are available on request from the corresponding authors.

Received: 6 February 2020; Accepted: 23 April 2020; Published: xx xx xxxx

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Acknowledgements Te authors thank Jiunn-Ren Roan for suggestions on the energetic perspective of mandible strikes; Wei-Sheng Wang, Ming-Huang Wu, and Ching-Lung Chiu (Utopia Instruments Co., Ltd.) for their assistance with the high-speed videography and analysis; and Wei-Ren Liang for collecting termites and providing suggestions on experimental methods. Tis work was supported by the Ministry of Science and Technology, Taiwan (MOST 102–2313-B-005-037-MY2 (H.-F.L.), MOST 104-2311-B-005-010-MY3 (K.-J.C.), and MOST 105-2815-C-005- 026-B (K.-C.K.)), and partially supported by the Ministry of Education–funded iEGG and Animal Biotechnology Center (MOE 107-S-0023-I (K.-J.C.)) of the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project. Author contributions K.-C.K., C.-I.C., K.-J.C. and H.-F.L. designed and performed the experiments. K.-C.K., C.-I.C. and H.-F.L. collected and identifed termites. K.-C.K., M.-C.S. and K.-J.C. conducted mechanical analyses of the snapping behaviour of soldier mandibles. K.-C.K., C.-I.C. and H.-F.L. designed the fgures. K.-C.K. and C.-I.C. wrote the manuscript in consultation with K.-J.C. and H.-F.L. All the authors discussed the study results.

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Competing interests Te authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66294-1. Correspondence and requests for materials should be addressed to K.-J.C. or H.-F.L. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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